Generation of a germ cell‐specific mouse transgenic Cre line, Vasa‐Cre

Abstract

SummaryCell type-specific genetic modification using the Cre/loxP system is a powerful tool for genetic analysis of distinct cell lineages. Because of the exquisite specificity of Vasa expression (confined to the germ cell lineage in invertebrate and vertebrate species), we hypothesized that a Vasa promoterdriven transgenic Cre line would prove useful for the germ cell lineage-specific inactivation of genes. Here we describe a transgenic mouse line, Vasa-Cre, where Cre is efficiently and specifically expressed in germ cells. Northern analysis showed that transgene expression was confined to the gonads. Cre-mediated recombination with the Rosa26-lacZ reporter was observed beginning at ~e15, and was >95% efficient in male and female germ cells by birth. There was no ectopic activity in most adults, although some animals showed more widespread lacZ expression. This Vasa-Cre transgenic line should thus prove useful for genetic analysis of diverse aspects of germ cell function and gametogenesis. KeywordsCre; vasa; germ cells; gonad Genetic approaches are well-suited for studies of gametogenesis and reproduction, processes that require complex interactions between germline and somatic cells, and hence are difficult to fully recapitulate in vitro. More recently, conditional gene targeting using the Cre/loxP system has emerged as an especially powerful method in reproductive genetics, particularly in the study of genes whose systemic inactivation is lethal. Critical to the success this approach is the availability of suitable Cre transgenes expressed in a wide range of distinct cell lineages at appropriate developmental timepoints (Kwan, 2002;Lewandoski, 2001). Previous germ cell Cre lines include ZP3-Cre, which is not active in primordial follicles but becomes induced following follicle growth; this line is thus useful for analyses of follicle growth or maternal contributions to the embryo (de Vries et al., 2000). A Cre knock-in into the TNAP (tissue nonspecific alkaline phosphatase) is active in primordial germ cells starting as early as e9.5. but the TNAP promoter is also active in other tissues (e.g. placenta and liver, among others) and such widespread expression may complicate interpretation of experiments where germ cellspecific gene inactivation is desired or required (if e.g. the locus is essential) (Lomeli et al., 2000).Vasa expression is confined to the germ cell lineage in species including Drosophila, zebrafish, mouse, and humans (Castrillon et al., 2000;Lasko and Ashburner, 1988 (Tanaka et al., 2000). We reasoned that a Vasa-Cre line could prove useful for studies of germ cell function following gonadal colonization, including many aspects of spermatogenesis and oogenesis, such as the assembly, activation, and growth of primordial follicles.We cloned a 5.6 kb DNA fragment containing mouse Vasa 5' regulatory sequences, including the transcriptional start site. This fragment contains canonical promoter sequences including a TATA box at −27 and more distant control elements important for germ cell specific ex...

Conditional inactivation of Miwi2 reveals that MIWI2 is only essential for prospermatogonial development in mice

“…To further explore potential roles of MIWI2 in the meiotic and haploid phases of spermatogenesis, we crossed Miwi2 loxp mice with three germ cell-specific Cre lines to inactivate Miwi2 in the male germ line at different developmental time points. The Ddx4-Cre line has been used to delete a floxed allele in PGCs/prospermatogonia with an onset of CRE expression at E15.5, 39 whereas Stra8-Cre mice display CRE expression in prospermatogonia at P3 although the full penetrance of CRE activity is not reached until pachytene spermatocytes at P12 10,40,41 ( Figure 1d). The Prm-Cre line has Cre expression starting in round spermatids, but the protein expression may be delayed due to posttranscriptional regulation 42 ( Figure 1d).…”

Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries

“…EGFP is constitutively expressed in all cells of Rosa26 rbw/+ and Rosa26 rbw/rbw mice. The Ddx4-Cre transgenic mice (33) were purchased from the Jackson Laboratory. To produce the Rosa26 rbw/+ ;Ddx4-Cre germline reporter mouse model, Rosa26 rbw/rbw females were crossed with Ddx4-Cre males.…”

Lin28 sustains early renal progenitors and induces Wilms tumor

“…Thus, we endeavored to overexpress Lin28 in PGCs by crossing mice containing a Lox-stop-Lox-Lin28a cassette (LSL-Lin28a) (Supplemental Fig. S1A) with mice carrying a Vasa-Cre transgene, which we anticipated would express Cre in PGCs when transmitted paternally, allowing us to test the potential for Lin28 to induce germ cell tumors (Gallardo et al 2007). Contrary to expectations, however, the cross between a LSL-Lin28a female and VasaCre male did not yield the predicted germ cell phenotype (zero out of 50) but unexpectedly produced renal tumors in 10% of the offspring (five out of 50; four bilateral and one unilateral) (Fig.…”

“…1B). Crosses of LSL-Lin28a males with females carrying the Vasa-Cre allele resulted in constitutional overexpression in all tissues by virtue of Cre expression in oocytes (Gallardo et al 2007) and perinatal lethality. Interestingly, the kidneys of transgenic embryonic day 18.5 (E18.5) embryos were larger than the kidneys of their littermate controls and contained fewer mature proximal tubules (Supplemental Fig.…”